Manumycin derivatives and the use thereof

ABSTRACT

##STR1## where R 1  is H or OH; 
     R 2  is H or OH; 
     R 3  is H, OH, OCH 3 , CH 3  or NH 2  ; 
     Rhu 4 is H, OH, OCH 3 , NH 2  or ##STR2## The compounds of the formula I are prepared by fermentation of the strain Streptomyces parvulus DSM 40722 in the presence of substituted benzoic acids of the general formula II ##STR3## where R 1  is H or OH; 
     R 2  is H or OH; 
     R 3  is H, OH, OCH 3 , CH 3  or NH 2  ; 
     R 4  is H, OH, OCH 3  or NH 2 , 
     and they inhibit Leukocyte elastase and can be used as pharmaceuticals.

DESCRIPTION

The present invention relates to manumycin derivatives of the generalformula I ##STR4## where R¹ is H or OH;

R² is H or OH;

R³ is H, OH, OCH₃, CH₃ or NH₂ ;

R⁴ is H, OH, OCH₃, NH₂ or ##STR5## The present invention also relates toa process for the preparation of compounds of the general formula I,which comprises fermentation of the strain Streptomyces parvulus Tu 64(DSM 40722) in the presence of substituted benzoic acids of the generalformula II ##STR6## where R¹ is H or OH;

R² is H or OH;

R³ is H, OH, OCH₃, CH₃ or NH₂ ;

R⁴ is H, OH, OCH₃ or NH₂,

in suitable nutrient media.

The compounds of the general formula I are derived from manumycin(formula III) which has been described by F. Buzzetti et al., (Phar.Acta. Helv., 1963, 38, 871) and whose structure and absoluteconfiguration have been determined by A. Zeeck et al. (J. Antibiotics,1987, 40, 1530 and 1549). ##STR7##

In order to avoid the normal biosynthesis of the central m-C₇ N starterunit for manumycin, non-physiological amounts of other C₇ precursormolecules were added to the culture medium during the stationary phaseof growth of Streptomyces parvulus (DSM 40722). Besides a carbon andnitrogen source, the abovementioned culture medium contains thecustomary inorganic salts. Extraction of the mycelium with an organicsolvent which is immiscible, or only slightly miscible, with waterresults in colored extracts from which compounds of the general formulaI are obtained after removal of the lipophilic constituents andpurification of the crude product by chromatography.

Hydrolysis of compounds of the general formula I with R⁴ = ##STR8##(side-chain of manumycin) with 20% strength methanolic KOH producescompounds of the general formula I with R⁴ =NH₂. The compounds (1) and(2) from Table 1 can also be obtained by reduction of manumycin (formulaIII) by known processes, such as described by A. Zeeck et al. (J.Antibiotics, 1987, 40, 1541). We have found that compounds of thegeneral formula I, especially the compounds (1), (2), (3) and (5) inTable 1, inhibit leukocyte elastase.

                                      TABLE 1                                     __________________________________________________________________________    Compounds of the general formula I with the                                   following radicals R.sup.1 to R.sup.4                                                 R.sup.1                                                                          R.sup.2                                                                         R.sup.3                                                                          R.sup.4                                                       __________________________________________________________________________    Compound (1)                                                                          OH H OH                                                                                ##STR9##                                                     Compound (2)                                                                          H  H OH                                                                                ##STR10##                                                    Compound (3) (= 64-mABA)                                                              H  H H                                                                                 ##STR11##                                                    Compound (4)                                                                          H  H H  NH.sub.2                                                      Compound (5)                                                                          H  H NH.sub.2                                                                         H                                                             (= 64-pABA)                                                                   __________________________________________________________________________

Leukocyte elastase, which is one of the neutral proteases, is involvedin the human or animal body in the degradation of a very wide variety ofsoluble and tissue-associated proteins.

Since, however, uncontrolled proteolytic activity would result in thedegradation of important regulatory proteins (such as those of thecoagulation and complement systems), under physiological conditions thebody maintains a balance between the enzyme and its naturally occurringinhibitors (such as, for example, α1-protease inhibitor,α2-macroglobulin). In various pathological states there may now be anincreased secretion of elastase from leukocytes (preferentially frompolymorphonuclear granulocytes) or a decreased release of inhibitors,and thus destruction of important regulatory or structural proteins.

The syndromes with this etiology are many and various and may affect avariety of organs and tissues; they include the various states of shock(for example septic shock), disturbances of the coagulation system, lungdisorders, such as acute respiratory distress syndrome (ARDS) andpulmonary emphysema, post-traumatic and postoperative complications,various types of inflammation, both in its acute and in its chronicform, such as rheumatoid arthritis and other collagenoses.

Furthermore, elastase secreted by tumor cells plays an important part inmetastasis and the invasive growth of tumors in that it destroys thebasement membrane surrounding the primary tumor and facilitates thepassage of tumor cells through this matrix.

Accordingly, the use of elastase inhibitors is of therapeutic benefit inall the disorders described.

The invention is explained in detail in the examples which follow.

EXAMPLE 1 Preparation of 64-mABA (Compound (3), see Table 1)

a) Preparation of a suspension of spores of the producer strain

100 ml of nutrient solution (4 g of yeast extract, 10 g of malt extract,4 g of glucose, 1 l of tapwater, pH before sterilization 7.3) in a 500ml Erlenmeyer flask with septum inlet are inoculated with the strain DSM40722 and incubated on a rotating shaker at 250 rpm at 28° C. for 72hours. Subsequently, 20 ml of culture liquid are uniformly distributedin a 500 ml Roux flask containing the nutrient medium of theabovementioned composition, to which 20 g of agar have been added perliter for solidification, and are decanted. The cultures are incubatedat 28° C. for 10 to 14 days. The spores which have been produced afterthis time in each Roux flask are rinsed out with 500 ml of deionizedwater, which contains one drop of a commercially available nonionicsurfactant, and are immediately used further or stored at -22° C.

b) Preparation of a culture or preculture of the producer strain in anErlenmeyer flask

A 500 ml Eerlenmeyer flask with septum inlet, containing 100 ml of anutrient solution composed of 2% defatted soybean meal, 2% mannitol andwater ad 100 ml (pH 7.5 before autoclaving) is inoculated with a culturegrown in a slant tube or with 0.2 ml of spore suspension, and isincubated on a shaker at 250 rpm at 28° C. Sufficient for theinoculation of 10, 20, 25 and 100 l fermenters is a 48-hour oldsubmerged culture (5%) from the same nutrient solution.

c) Fermentation

A fermenter of 15 l capacity is operated as follows. 4 l of air perminute are passed into the culture liquid at an incubation temperatureof 28° C. and at 250 rotations per minute (medium: defatted soybean meal2%, mannitol 2%, pH of the solution adjusted to 7.5 with NaOH beforesterilization). Foam formation can be suppressed by repeated addition ofa few drops of ethanolic polyol solution. After 30-45 hours, preferably36 hours, 55 mmol of 3-aminobenzoic acid (dissolved in a little water,pH=7) per liter of culture medium are added. After a further 38 hours,the culture is adjusted to pH 4.5 with 2M HCl, stirred with Hyflo Celite(50 g per liter) for 10 minutes and filtered. The filter residue isextracted three times with acetone (250 ml per liter of culturesolution), and the culture filtrate is extracted three times with ethylacetate: the extracts are concentrated. The aqueous-oily residues arecombined and extracted four times with chloroform (30 ml per liter ofculture solution). The combined organic phase yield, after evaporationof the solvent, an oily crude product which solidifies on treatment withice-cold petroleum ether. The dark brown amorphous powder ischromatographed twice on Sephadex LH-20 columns (100×2.5 cm, agent forapplication and elution: CHCl₃). This is followed by chromatography onan RP-8 silica gel column (acetonitrile/water, 3:1 v/v) in order toyield, after an optimal fermentation, 123 mg/l of culture broth of pureyellow amorphous 64-mABA (compound (3)). The melting point is 196°-197°C.

Other properties are as follows

[α]_(D) ²² is -213° (c=0.15 in CHCl₃) IR (KBr): 3420, 3260, 2940, 2910,1605, 1575(sh) 1530 and 996 cm⁻¹

UV spectrum: λ_(max) (Methanol)=351 (40 800), 263 (25 100)

¹³ C, 1H-NMR (200 MHz, CDCl₃): _(c) =197.4 (s, C-1"), 174.3 (C-3"),168.7 (s, C-1'), 165.9 (s, C-13), 144.0 (d, C-11), 142.1 (d, C-5'),141.6 (d, C-9), 138.9 (d, C-3'), 138.8 (s, C-2), 137.4 (s, C-4), 136.9(d, C-7), 130.2 (d, C-10), 130.1 (s, C-2'), 129.4 (s, C-4'), 129.3 (d,C-6), 128.5 (d, C-8), 123.0 (d, C-5), 120.4 (d, C-12), 120.1 (d, C-1),118.0 (d, C-3), 115.3 (s, C-2"), 37.1 (t, C-7'), 32.8 (d, C-6'), 32.2(t, C-5"), 29.8 (t, C-8' ), 25.7 (t, C-4"), 22.8 (t, C-9'), 20.8 (q,C-13'), 16.6 (q, C-12'), 14.4 (q, C-10'), 14.1 (q, C-11'), δ_(H)(CDC1₃), 0.86 (t, H 6.4 Hz, 10'-H₃), 0.93 (d, J 6.4 Hz, 13'-H₃),1.14-1.42 (m, broad, 7'-H₂, 8'-H₂, 9'-H₂), 1.84 (d, j 1.6 Hz, 12'-H₃),2.12 (d, j 1.6 Hz, 11'-H₃), 2.34-2.50 (m, broad, 6'-H), 2.58-2.60 (s,broad, 4"-H₂, 5"-H₂), 5.36 (d, j 10 Hz, 5'-H), 6.09 (d, j 15 Hz, 12-H),6.42 (dd, j 13.5 and 12 Hz, 10-H), 6.62-6.88 (m, 7-H, 9-H, including6.82, 3'-H), 6.90 (dd, j 15.5 and 11 Hz, 8-H), 7.15 (d, broad j 7.5 Hz,5-H), 7.22-7.46 (m, 1-H, 11-H), 11-H), 7.64 (s, broad, NH), 7.86 (s,3-H), 7.88 (s, broad, NH), 13.80 (s, broad, OH);

Mass spectroscopy: m/z 502. (21%, M⁺, found: 502.2832 for C₃₁ H₃₈ N₂ O₄)417 (24%), 324 (11%), 193 (38%), 123 (11%), 109 (64%).

Elemental analysis: C, 74.04; H, 7.81; N, 5.49. C₃₁ H₃₈ N₂ O₄ requiresC, 74.06; H, 7.62, N, 5.57.

EXAMPLE 2 Preparation of Compound (4) in Table 1 by Hydrolysis of64-mABA

94 mg of 64-mABA were dissolved in 50 g of 20% strength methanolic KOHand refluxed for 24 hours. The yellow reaction mixture was adjusted topH 2 with aqueous oxalic acid and extracted twice with 300 ml of CHCl₃.The combined organic extracts were dried with Na₂ SO₄ and evaporated,resulting in a yellow amorphous powder. This crude product yielded aftercolumn chromatography on silica gel (45 cm×2.5 cm, CHCl₃ /MeOH, 9:1 v/v)three main products:

(a) 5.8 mg starting material,

RF=0.60 (CHCl₃ /MeOH, 9:1 v/v)

(b) 30 mg pale yellow oil,

RF=0.43 (CHCl₃ /MeOH, 9:1 v/v )

(c) 34.2 mg yellow powder,

RF=0.39 (CHCl₃ /MeOH, 9:1 v/v )

(b) yielded after further purification on a Sephadex LH-20 column (45cm×2.5 cm, CHCl₃) 20.0 mg (54%) of 2,4,6-trimethyl-2,4-decadienoic acid(formula IV) ##STR12##

Product (c) yielded after chromatography on a Sephadex LH-20 column (50cm×2.5 cm, CHCl₃) 302 mg (55%) of compound (4) as a yellow amorphouspowder with the following properties:

Melting point: 256° C.

IR (KBr): 3440, 3380, 3260, 1680 (sh) 1610, 1550 and 1008 cm⁻¹.

UV spectrum in methanol: λ_(max) =342 (44 500), 259 (24 600).

¹³ C, ¹ H-NMR (200 MHz, DMSO-d₆): .sup.δ_(H) 2.09 (s, 4"-H₂), 2.48 (s,5"-H₂), 3.30 (s, broad, NH, overlapped by HOD), 6.43-7.08 (m, 9protons), 7.29 (dd, J 15.0 and 12.5 Hz, 11-H), 991 (s, broad, OH);.sup.δ_(C) 166.1 (s, C-13), 148.8 (s, C-2), 142.5 (d, C-11), 137.3 (d,C-9), 136.9 (d, C-7), 129.9 (s, C-4), 114.8 (s, C-2"), 28.8 (t, broad,C-4"), C-5"), the signals 129.1 (d), 127.4 (d), 121.2 (d), 119.0 (d),114.9 (d), 114.6 (d) and 112.5 (d) could not be assigned unambiguouslyto C-1, C-3, C-5, C-6, C-8, C-10 and C-12, and the signals for C-1" andC-3" were not observed.

Mass spectrometry: m/z 310 (13%, M⁺, found: 310.1317 for C₁₈ H₁₈ N₂ O₃),198 (49%), 180 (15%), 170 (92%), 132 (100%).

EXAMPLE 3 Preparation of 64-pABA (Compound (5) in Table 1)

55 mmol/liter 4-aminobenzoic acid were fed to a 36-hour old culture ofStreptomyces parvulus DSM 40722 (conditions as in 1c). The extractionsof the mycelium and culture filtrate after 74 hours were carried out asdescribed above. The dark brown crude product yielded after columnchromatography twice on silica gel (30 cm×5 cm, CHCl₃ /MeOH, 9:1 v/v)35.8 mg/liter 64-pABA (compound (5)) as a yellow amorphous powder withthe following properties:

Melting point: 242° C.

IR (KBr): 3440, 3350, 3240, 2930, 2860, 1710 sh, 1615 sh, 1585, 1000cm⁻¹.

UV spectrum in methanol: 392 (31 500), 261 (14 400), 201 (15 200) ¹³ C,¹ H NM(200 MHz, DMSO-d₆): .sup.δ C 166.4 (s, C-13), 149.6 (s, C-1),143.1 (d, C-11), 142.5 (d, C-9), 137.8 (d, C-7), 128.3 (d, C-3 and C-5),127.3 (d, C-10), 124.1 (s, C-4), 122.9 (d, C-8), 119.4 (d, C-12), 114.9(s, C-2"), 113.8 (d, C-2 and C-6), 28.8 (t, broad, C-4" and C-5"), thesignals for C-1" and C-3" could not be observed.

.sup.δ_(H) 2.10 (s, 4"-H₂), 2.47 (s, 5"-H₂), 3.30 (s, broad, NH,overlapped by HOD), 5.50 (s, broad, NH), 6.34-6.94 (m, 7 protons,including 6.57 (d, J 8.5 Hz, 2-H and 6-H)), 7.23 (d, J 8.5 Hz, 3-H and5-H), 7.30 (dd, J 14.5 and 12 Hz, 11-H), 9.94 (s, broad, OH);

Mass spectrometry: m/z 310 (8.4% M⁺ Found: 310.1317 for C₁₈ H₁₈ N₂ O₃)198 (10.4%), 170 (40.7%), 132 (100%).

EXAMPLE 4 Assay for Elastase from Polymorphonuclear Granulocytes (PMNElastase)

The elastase was isolated from human leukocytes by the method describedby Engelbrecht et al. (Hoppe-Seyler's Physiol. Chem. 363, pp. 305-315,1982). The substrate used was the MeO-Suc-Ala-Ala-Pro-Val-pNA(Calbiochem) described by Nakajima et al. (J. Biol. Chem. 254, pp.4027-4032, 1979). The Liberation of the p-nitroaniline from thesubstrate in 15 minutes was measured as the increase in absorption at405 nm in a spectrophotometer. This absorption was defined as 100%activity of the enzyme PMN elastase. The inhibitors of PMN elastase werepreincubated in increasing concentrations up to a maximum of 100 μg/mlwith the enzyme for 1 hour, and the enzyme reaction was started with thesubstrate. The IC₅₀ was defined as that inhibitor concentration whichinhibits 50% of the enzyme activity. Substances which showed noinhibiting effect at the maximum concentration of 100 μg/ml were definedas inactive.

The IC₅₀ values obtained are compiled in Tab. 2. The activity of thereference compound manumycin is comparable to that of the compounds (1)to (3), whereas compound (5) has a distinctly weaker inhibiting effect.Compound (4) is inactive in this assay.

                  TABLE 2                                                         ______________________________________                                        Inhibiting effect of manumycin and the tested                                 manumycin derivatives on PMN elastase                                         Substance           IC.sub.50 (μg/ml)                                      ______________________________________                                        Manumycin (formula III)                                                                           4.4                                                       Compound (1)        2.5                                                       Compound (2)        3.1                                                       Compound (3) (= 64-mABA)                                                                          3.6                                                       Compound (4)        --                                                        Compound (5) (= 64-pABA)                                                                          42.0                                                      ______________________________________                                    

We claim:
 1. A compound of the formula I ##STR13## where R¹ is H orOH;R² is H or OH; R³ is H, OH, OCH₃, CH₃ or NH₂ ; R⁴ is H, OH, OCH₃, NH₂or ##STR14## with the exception of compound (1) with R₁ =OH, R₂ =H, R₃=OH and ##STR15## and of compound (2) with R₁ =H, R₂ =H, R₃ =OH and##STR16## and of compound (4) with R₁ =R₂ =R₃ =H, and R₄ is NH₂.
 2. Acompound of the formula I, where R₁ =R₂ =R₃ =H, and R⁴ is ##STR17##
 3. Acompound of the formula I, where R₁ =R₂ =R₄ =H, and R₃ is NH₂.
 4. Apharmaceutical composition for the inhibition of elastase comprising aneffective amount of a compound of formula I as claimed in claim 1 or asalt thereof together with a pharmaceutically acceptable carrier.
 5. Amethod for inhibiting elastase comprising contacting said elastase witha compound of formula I as claimed in claim 1.